1. Field
The present disclosure relates to methods of amplifying a target nucleic acid and methods of determining a relative amount of a target nucleic acid in a sample.
2. Description of the Related Art
Nucleic acid amplification refers to an increase in the number of copies of a target nucleic acid sequence or its complementary sequence. Nucleic acid amplification is well known in the art. The amplification of a nucleic acid includes methods that require multiple cycles during the amplification process, and methods that are performed at a single temperature. Cycling techniques are exemplified by methods requiring thermo-cycling, such as polymerase chain reaction (PCR). PCR generally involves thermally denaturing a double-stranded DNA to provide single stranded DNAs, annealing a primer to the single stranded DNA; and synthesizing a complementary strand from the primer.
Isothermal amplification is an amplification process performed at a single temperature, or wherein the major aspect of the amplification process is performed at a single temperature. In the PCR process, the product of the reaction is heated to separate the two strands of DNA such that a primer may bind to the template strand. Conversely, the isothermal techniques rely on a strand displacing polymerase in order to separate the two strands of a double strand and re-copy the template. Isothermal techniques may be classified into methods that rely on the replacement of a primer to initiate a reiterative template copying, and those that rely on continued re-use or new synthesis of a single primer molecule. The methods that rely on the replacement of the primer include helicase dependant amplification (HDA), exonuclease dependant amplification, recombinase polymerase amplification (RPA), and loop mediated amplification (LAMP). The methods that rely on continued re-use or new synthesis of a single primer molecule include strand displacement amplification (SDA) or nucleic acid based amplification (NASBA and TMA).
The above methods can be used to amplify short nucleic acid sequences, such as microRNAs (miRNAs). However, an amplification bias often is generated, which will vary according to the number of copies of the target nucleic acid and relative abundance of certain nucleotides, for example, AT content or GC content. For example, when each of microRNAs (miRNAs) is amplified in a sample including a plurality of miRNAs, i.e., in a sample including a miRNA library, the amplification bias may vary according to the sequence of the miRNAs and the number of copies of the miRNAs present in the sample. In general, miRNA has a length in the range of about 18 to 25 nucleotides (nt) and about 21 to 24 nt on average and has more than 1000 types of sequences.
Thus, there is still a need to develop a method of amplifying a plurality of target nucleic acids, particularly short nucleic acid sequences such as an miRNA library, without amplification bias or with reduced amplification bias.